Clostridium difficile causes over 500,000 cases of infections per year, with an estimated 15,000 deaths and a conservatively estimated economic cost at $1-3 billion in the US annually. Currently, standard treatment for C. difficile infection (CDI) is the administration of one of several antibiotics which include metronidazole, vancomycin or the newly developed fidaxomicin. While effective, these treatments have a disease recurrence rate of 15-35%, due to their disruptive impact on the gut microbiome. More effective strategies to treat primary and recurrent CDI are urgently needed. No vaccine is currently licensed against CDI. The objective of this project is to develop novel vaccines that target both C. difficile colonization/growth factors and C. difficile toxins. The symptoms of CDI are attributed to liberation of two C. difficile toxins, TcdA and TcdB. We recently constructed a recombinant fusion protein, mTcd138, which contains the immunodominant regions of TcdA and TcdB. Since spores are the major cause of disease transmission and recurrence, we propose to enhance our prototype vaccine by including additional virulence factor antigens to reduce or eliminate the excretion of spores. Decreasing spore excretion will reduce or eliminate the risk of disease recurrence and transmission. In Aim 1 we will first construct fusion immunogens containing mTcd138 and major C. difficile colonization factors, and then determine the immunogenicity and protection against CDI and C. difficile spore colonization in mice immunized intramuscularly or sublingually with the fusion immunogens. Mucosal is often more effective against enteric pathogens such as C. difficile. In Aim 2 we will develop a mucosal/oral vaccine by expressing the chosen vaccine candidate from Aim1 in the Bacillus subtilis mucosal delivery system.